Repurposing bromocriptine for Aβ metabolism in Alzheimer’s disease (REBRAnD) study : randomised placebo-controlled double-blind comparative trial and open-label extension trial to investigate the safety and efficacy of bromocriptine in Alzheimer’s disease with presenilin 1 (PSEN1) mutations

Introduction Alzheimer’s disease (AD) is one of the most common causes of dementia. Pathogenic variants in the presenilin 1 (PSEN1) gene are the most frequent cause of early-onset AD. Medications for patients with AD bearing PSEN1 mutation (PSEN1-AD) are limited to symptomatic therapies and no established radical treatments are available. Induced pluripotent stem cell (iPSC)-based drug repurposing identified bromocriptine as a therapeutic candidate for PSEN1-AD. In this study, we used an enrichment strategy with iPSCs to select the study population, and we will investigate the safety and efficacy of an orally administered dose of bromocriptine in patients with PSEN1-AD. Methods and analysis This is a multicentre, randomised, placebo-controlled trial. AD patients with PSEN1 mutations and a Mini Mental State Examination-Japanese score of ≤25 will be randomly assigned, at a 2:1 ratio, to the trial drug or placebo group (≥4 patients in TW-012R and ≥2 patients in placebo). This clinical trial consists of a screening period, double-blind phase (9 months) and extension phase (3 months). The double-blind phase for evaluating the efficacy and safety is composed of the low-dose maintenance period (10 mg/day), high-dose maintenance period (22.5 mg/day) and tapering period of the trial drug. Additionally, there is an open-labelled active drug extension period for evaluating long-term safety. Primary outcomes are safety and efficacy in cognitive and psychological function. Also, exploratory investigations for the efficacy of bromocriptine by neurological scores and biomarkers will be conducted. Ethics and dissemination The proposed trial is conducted according to the Declaration of Helsinki, and was approved by the Institutional Review Board (K070). The study results are expected to be disseminated at international or national conferences and published in international journals following the peer-review process

cGMP dynamics underlie thermosensation in C. elegans

Animals sense ambient temperature so that they can adjust their behavior to the environment; they avoid noxious heat and coldness and stay within a survivable temperature range. C. elegans can sense temperature, memorize past cultivation temperature and navigate towards preferable temperature, for which a thermosensory neuron, AFD, is essential. AFD responds to temperature increase from the past cultivation temperature by increasing intracellular Ca2+ level. We aimed to reveal how AFD encodes and memorizes the information of temperature. Although cGMP synthesis is crucial for thermosensation by AFD, whether and how cGMP level temporally fluctuates in AFD remained elusive. We therefore monitored cGMP level in AFD and found that cGMP dynamically responded to temperature change in a manner dependent on past cultivation temperature. Given that cGMP dynamics is supposed to be upstream of Ca2+ dynamics, our results suggest that AFD’s memory is formed by simpler molecular mechanisms than previously expected from the Ca2+ dynamics. Moreover, we analyzed how guanylyl cyclases and phosphodiesterases, which synthesize and degrade cGMP, respectively, contributed to cGMP and Ca2+ dynamics and thermotaxis behavior

cGMP dynamics that underlies thermosensation in temperature-sensing neuron regulates thermotaxis behavior in C. elegans

Living organisms including bacteria, plants and animals sense ambient temperature so that they can avoid noxious temperature or adapt to new environmental temperature. A nematode C. elegans can sense innocuous temperature, and navigate themselves towards memorize past cultivation temperature (Tc) of their preference. For this thermotaxis, AFD thermosensory neuron is pivotal, which stereotypically responds to warming by increasing intracellular Ca2+ level in a manner dependent on the remembered past Tc. We aimed to reveal how AFD encodes the information of temperature into neural activities. cGMP synthesis in AFD is crucial for thermosensation in AFD and thermotaxis behavior. Here we characterized the dynamic change of cGMP level in AFD by imaging animals expressing a fluorescence resonance energy transfer (FRET)-based cGMP probe specifically in AFD and found that cGMP dynamically responded to both warming and cooling in a manner dependent on past Tc. Moreover, we characterized mutant animals that lack guanylyl cyclases (GCYs) or phosphodiesterases (PDEs), which synthesize and hydrolyze cGMP, respectively, and uncovered how GCYs and PDEs contribute to cGMP and Ca2+ dynamics in AFD and to thermotaxis behavior

cGMP dynamics that underlies thermosensation in temperature-sensing neuron regulates thermotaxis behavior in C. elegans.

Living organisms including bacteria, plants and animals sense ambient temperature so that they can avoid noxious temperature or adapt to new environmental temperature. A nematode C. elegans can sense innocuous temperature, and navigate themselves towards memorize past cultivation temperature (Tc) of their preference. For this thermotaxis, AFD thermosensory neuron is pivotal, which stereotypically responds to warming by increasing intracellular Ca2+ level in a manner dependent on the remembered past Tc. We aimed to reveal how AFD encodes the information of temperature into neural activities. cGMP synthesis in AFD is crucial for thermosensation in AFD and thermotaxis behavior. Here we characterized the dynamic change of cGMP level in AFD by imaging animals expressing a fluorescence resonance energy transfer (FRET)-based cGMP probe specifically in AFD and found that cGMP dynamically responded to both warming and cooling in a manner dependent on past Tc. Moreover, we characterized mutant animals that lack guanylyl cyclases (GCYs) or phosphodiesterases (PDEs), which synthesize and hydrolyze cGMP, respectively, and uncovered how GCYs and PDEs contribute to cGMP and Ca2+ dynamics in AFD and to thermotaxis behavior

Strain list.

(DOCX)</p

<i>pde-1</i> and <i>pde-2</i> act in AFD to regulate thermotaxis.

A. Wild type, pde-1, pde-2 and pde-1; pde-2 animals and pde-1; pde-2 animals that express PDE-1 or PDE-2 specifically in AFD were cultivated at 17°C or 23°C and then subjected to thermotaxis assay. n = 8 for N2 and pde-1; pde-2. n = 4 for others. The error bars in histograms represent the standard error of mean (SEM). The thermotaxis indices of strains marked with distinct alphabets differ significantly (p pde gene(s) indicated that express cGi-500 cGMP indicator in AFD were cultivated at 23°C and subjected to imaging analysis. Warming and cooling was at the rate of 1°C/6 sec. Individual (gray) and average (blue) fluorescence ratio (CFP/YFP) change at AFD sensory ending is shown. C-D. Wild type and mutant animals lacking pde gene(s) indicated that express GCaMP3 Ca2+ indicator and tagRFP in AFD were cultivated at 23°C and subjected to imaging analysis. Warming and cooling was at the rate of 1°C/20 sec. Individual (gray) and average (pea green or green) fluorescence ratio (GCaMP/RFP) change at AFD sensory ending (C) and soma (D) is shown. (TIF)</p

Contribution of GCYs and PDEs on cGMP and Ca²⁺ dynamics in AFD cultivated at different temperature.

In animals cultivated at 17°C, any of GCY-23, GCY-8 and GCY-18 can probably contribute to the cGMP increment in response to warming since all of three gcy double mutants show Ca2+ response (S2 and S3 Figs). cGMP production by GCY-18 alone might not be sufficient since gcy-23 gcy-8 shows slightly defective thermotaxis (Fig 2A). Ca2+ level in soma is actively decreased via the three GCYs (S3 Fig) and SLO-2 potassium channel [40]. In animals cultivated at 23°C, activity of GCY-23 and GCY-8 is suppressed below the threshold temperature by coexistence of both, possibly forming an inactive dimer. Threshold for GCY-18 seems to be adjustable by an unknow AFD-specific mechanism (See ’Discussion‘ section). Transcription of gcy-18 is increased under higher cultivation temperature [40, 41]. Transcription of gcy-8 might be regulated by GCY-18 and TAX-4 (S4A Fig). Importantly, PDE-5 and PDE-1 collaborate to suppress Ca2+ level under threshold temperature, which seems to be essential for thermotaxis.</p

Ca²⁺ onsets from lower temperature in AFD soma of <i>gcy</i> double mutants (related to Fig 2).

Wild type and gcy double mutant animals indicated that express GCaMP3 Ca2+ indicator and tagRFP in AFD were cultivated at 17°C (left) or 23°C (right) and subjected to imaging analysis with temperature stimuli indicated (orange line). Warming and cooling was at the rate of 1°C/20 sec. n = 4 to 6. Individual (gray) and average fluorescence ratio (GCaMP/RFP) change at AFD soma is shown. B. Temperature at which Ca2+ level started increasing in response to warming was extracted using a MATLAB command ‘findchangepts’ and plotted. p values were indicated, or *** indicates p (TIF)</p

Ca²⁺ onsets from lower temperature in AFD sensory ending of gcy double mutants (related to Fig 2).

Wild type and gcy double mutant animals indicated that express GCaMP3 Ca2+ indicator and tagRFP in AFD were cultivated at 17°C (left) or 23°C (right) and subjected to imaging analysis with temperature stimuli indicated (orange line). Warming and cooling was at the rate of 1°C/20 sec. n = 4 to 6. Individual (gray) and average fluorescence ratio (GCaMP/RFP) change at AFD sensory ending is shown. B. Temperature at which Ca2+ level started increasing in response to warming was extracted using a MATLAB command ‘findchangepts’ and plotted. p values were indicated, or *** indicates p (TIF)</p